Uses of 2-bromopalmitate in the treatment of autoimmune disease

ABSTRACT

The present invention provides a method of inhibiting Fyn/Lck fatty acylation and protein palmitoylation in a cell in an individual in need of such treatment comprising the step of administering to said individual a pharmacologically effective dose of 2-bromopalmitate. Also provided is a method of treating an individual having an autoimmune disease comprising the step of administering to said individual a pharmacologically effective dose of 2-bromopalmitate.

CROSS REFERENCE TO RELATED APPLICATION

This is a 371 of PCT/US00/26190 filed Sep. 22, 2000 and claims benefitof priority of U.S. Provisional Application No. 60/155,743, filed Sep.23, 1999, now abandoned.

FEDERAL FUNDING LEGEND

This invention was produced in part using funds obtained through grantsGM57966 and CA29502 from the National Institute of Health. Consequently,the federal government has certain rights in this invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the fields of the molecularbiology of T cell signaling and fatty acid biochemistry andpharmacology. More specifically, the present invention relates to noveluses of 2-bromopalmitate.

2. Description of the Related Art

Many viral and cellular proteins are modified by fatty acid acylationwith myristate or palmitate (1,2). For example, all members of the Srcfamily of tyrosine protein kinases are covalently modified by the 14carbon fatty acid myristate. Myristate is co-translationally attached toa glycine at position 2 of the protein through an amide linkage, in aprocess catalyzed by N-myristoyl transferase (NMT) (35). Myristoylationhas been shown to be necessary (6,7) but not sufficient (8) for membranebinding. In addition, all Src proteins use a second membrane targetingsignal. For seven out of the nine Src family members, this second signalinvolves modification with the 16 carbon fatty acid palmitate. Palmitateis post-translationally attached to a cysteine residue within anN-terminal myr-gly-cys consensus motif (9).

Attachment of myristate and palmitate to Src family kinases enhances thelocalization of these proteins to the plasma membrane, where they mustbe present in order to function properly. In addition, proteinpalmitoylation has been shown to be critical for localization ofproteins to specialized subdomains of the plasma membrane that areresistant to detergent extraction (10-15). These detergent resistantmicrodomains (detergent resistant microdomains), also known as rafts,are enriched in cholesterol, glycosphingolipids, and GPI-anchoredproteins (16-18). Localization to detergent resistant microdomainsinfluences the ability of key signaling molecules to interact with eachother and to participate in signaling from the cell surface to theinterior of the cell (11,19-21).

The importance of protein fatty acylation is best illustrated 0.5 in Tcell receptor (TCR) mediated signal transduction. The Src relatedkinases Fyn and Lck are highly expressed in cells of hematopoieticorigin, particularly lymphocytes (22), and are required for signalingthrough the T cell receptor. Protein tyrosine phosphorylation is one ofthe first events that occurs after binding of antigens to surfacereceptors in T lymphocytes. Upon receptor engagement, Fyn and Lckphosphorylate tyrosine residues found within multiple immunoreceptortyrosine-based activation motifs (ITAMS) located on the cytosolicportions of the TCRζ and CD3 chains. Immunoreceptor tyrosine-basedactivation motifs phosphorylation recruits key molecules that mediatedownstream signaling, including the tyrosine kinase ZAP-70 (19). One ofthe targets for activated ZAP-70 is LAT, a palmitoylated transmembraneprotein (10). Several recent studies have established that the abilityof Lck, Fyn and LAT to function in T cell receptor-mediated signalingdepends on their fatty acylation and localization to detergent resistantmicrodomains. Palmitoylation of Lck was shown to be essential for itssignaling function in T lymphocytes (11). Fyn must be palmitoylated andlocalized to detergent resistant microdomains in order to interact withthe ζ chain of the T cell receptor (23). Moreover, LAT must bepalmitoylated and in detergent resistant microdomains in order to becometyrosine phosphorylated and participate in downstream signaling (20).

To date, studies of the role of protein palmitoylation in variouscellular pathways have suffered from two major drawbacks. First, incontrast to N-myristoylation, very little is known about the enzymologyand biochemistry of protein palmitoylation. Two thioesterases, PPT1 andAP1, have been identified that deacylate palmitoylated Ras and Gαproteins in vitro (24,25). However, the enzyme(s) that catalyze(s)attachment of palmitate to proteins have not been definitivelyidentified. Several recent studies have described purification ofpalmitoyl acyl transferase (PAT) activities (26-28), while other reportshave documented that non-enzymatic palmitoylation can occur undercertain conditions in vitro (29,30). Second, nearly all studies reportedto date on the role of palmitoylation in cellular functions have beenlimited to expressing non-acylated mutant forms of proteins in varioussystems (11,20). While this approach does provide useful information, itis limited by the need to overexpress the mutant proteins. Furthermore,the loss of a cysteine residue, and not the loss of palmitate per se,may impair the ability of the protein to function properly. For example,Hepler et al showed that cysteine residues at the amino terminus of theGq alpha subunit is important for its interaction with effector andreceptor molecules, regardless of their state of palmitoylation (31).

Polyunsaturated fatty acids (PUFAs), particularly the n-3 series, areused clinically as immunosuppressive agents (32) and in the treatment ofvarious inflammatory diseases (33-36). Recently, it was reported thatpolyunsaturated fatty acids inhibit T cell signal transduction bydisplacing Fyn and Lck from the detergent resistant microdomains (37).The inhibitory effects of polyunsaturated fatty acids were hypothesizedto be mediated by modification of DRM structure and composition.

The prior art is deficient in the lack of specific inhibitors of Fyn andLck fatty acylation and protein palmitoylation. The present inventionfulfills this longstanding need and desire in the art.

SUMMARY OF THE INVENTION

The present invention documents the discovery of 2-bromopalmitate as aninhibitor of Fyn/Lck fatty acylation in general, and palmitoylation inparticular. 2-bromopalmitate preferentially blocks palmitoylation ofN-terminally palmitoylated proteins, and inhibits membrane binding andlocalization of Fyn to detergent resistant microdomains in COS-1 cells.Moreover, treatment of Jurkat T cells with 2-bromopalmitate partiallyblocks localization of endogenous Fyn, Lck and LAT to rafts, andinhibits T cell receptor-mediated signaling events including enhancedtyrosine phosphorylation, calcium flux and activation of MAP kinase. Theidentification of 2-bromopalmitate as an inhibitor of fatty acylation ofSrc family kinases serves to provide insight into the role of proteinpalmitoylation in Src mediated signal tranduction pathways.

The present invention also demonstrates that polyunsaturated fatty acidsare inhibitors of Fyn palmitoylation, and discloses a novel mechanism ofaction by which these agents exert their immunosuppressive effects.

In one embodiment of the present invention, there is provided a methodof inhibiting Fyn/Lck fatty acylation and protein palmitoylation in acell in an individual in need of such treatment comprising the step ofadministering to said individual a pharmacologically effective dose of2-bromopalmitate.

In another embodiment of the present invention, there is provided amethod of treating an individual having a pathophysiological statecomprising the step of administering to said individual apharmacologically effective dose of 2-bromopalmitate.

In yet another embodiment of the present invention, there is provided apharmaceutical composition comprising 2-bromopalmitate and apharmaceutically acceptable carrier.

Other and further aspects, features, and advantages of the presentinvention will be apparent from the following description of thepresently preferred embodiments of the invention given for the purposeof disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

So that the matter in which the above-recited features, advantages andobjects of the invention, as well as others which will become clear, areattained and can be understood in detail, more particular descriptionsof the invention briefly summarized above may be had by reference tocertain embodiments thereof which are illustrated in the appendeddrawings. These drawings form a part of the specification. It is to benoted, however, that the appended drawings illustrate preferredembodiments of the invention and therefore are not to be consideredlimiting in their scope.

FIG. 1 shows the effect of 2-bromopalmitate on Fyn fatty acylation andsubcellular localization in COS-1 cells. Transfected cells werepreincubated overnight with 100 μM 2-bromopalmitate, as described below.FIG. 1A: cells were radiolabeled for 4 hours in the absence (C) orpresence (2BP) of 2-bromopalmitate with 125I-IC13 or 125I-IC16 (toppanel), or with Tran35S-label (bottom panel), lysed and subjected toimmunoprecipitation with anti-Fyn antibody. Lysates were subjected toSDS-PAGE followed by phosphorimaging. FIG. 1B: cells were radiolabeledwith Tran³⁵S-label for 5 minutes, fractionated into particulate P100 (P)fractions and soluble S100 (S) fractions by centrifugation at 100,000×g,and subjected to immunoprecipitation, SDS-PAGE and phosphorimaging. FIG.1C: cells were lysed in buffer containing 1% Triton X-100. Detergentsoluble (S) and resistant (R) fractions were clarified at 100,000×g andsubjected to immunoprecipitation and SDS-PAGE followed by immunoblottingwith anti Fyn antibodies.

FIG. 2 shows the effect of 2-bromopalmitate on localization ofFyn(16)-eGFP in COS-1 cells. FIG. 2A: cells transiently expressingFyn(16)-eGFP were preincubated overnight in the absence (top) orpresence of 100 μM 2-bromopalmitate (middle) or 100 μM2-hydroxymyristate (bottom) and examined live by fluorescencemicroscopy. FIG. 2B: duplicates of the above samples were subjected tosubcellular fractionation into P100 and S100 fractions and subjected toimmunoblotting with anti-GFP antibody. 2BP: 2-bromopalmitate. 2OH myr: 2hydroxymyristate.

FIG. 3 shows the effect of 2-bromopalmitate on subcellular localizationof palmitoylated proteins. COS-1 cells were transiently transfected andtreated as follows: FIG. 3A: GαO(10)Fyn. Cells were treated overnightwithout (C) or with (2BP) 2-bromopalmitate, labeled for 5 minutes withTran35S-label and subjected to cellular fractionation into P100 (P) andS100 (S) fractions, followed by immunoprecipitation with anti-Fynantibody, SDS-PAGE and phosphorimaging. FIG. 3B: GAP43(10)-Fyn. Cellswere treated with or without 2-bromopalmitate as in (FIG. 3A) with theexception of labeling for 2 hours, to allow newly synthesized protein toreach the plasma membrane. FIG. 3C: G2A,C3SFyn-HRas. Cells were treatedwith 2-bromopalmitate as in (FIG. 3A). After fractionation, lysates weresubjected to immunoprecipitation followed by immunoblotting withanti-Fyn antibodies. FIG. 3D: G12V-Hras. Cells were treated with orwithout 2-bromopalmitate as in (FIG. 3A) and fractionated, following byimmunoblotting with anti Ras antibody. The faster migrating bandrepresents processed Ras. The slower migrating band (( ) representsunprocessed, cytosolic Ras).

FIG. 4 shows the effect of 2-bromopalmitate on Fyn fatty acylation andDRM localization of palmitoylated proteins in Jurkat T cells. Cells weretransfected by electroporation a nd preincubated for 3 hours with 100 μM2-bromopalmitate, as described below. FIG. 4A: cells were radiolabeledfor 4 hours in the absence (C) or presence (2BP) of 2-bromopalmitatewith ¹²⁵I-IC13 or ¹²⁵I-IC16 (top panel). Lysates were immunoprecipitatedwith anti-Fyn antibody. Bottom panel: to monitor total protein levels,aliquots from each sample were subjected to immunobloting with anti-Fynantibody. FIG. 4B: Localization to detergent resistant microdomains.Cells were cultured with 2-bromopalmitate as in (FIG. 4A), lysed withbuffer containing 0.5% Triton X-100, and layered on the bottom of asucrose gradient as detailed below. After overnight centrifugation, 1 mlfractions were collected and the DRM localization of endogenouspalmitoylated proteins was analyzed by SDS-PAGE and immunoblotting withanti-Lck (top), anti-Fyn (middle) or anti-LAT (bottom) antibodies.

FIG. 5 shows tyrosine phosphorylation of signaling proteins in Jurkat Tcells. FIG. 5A: Jurkat Cells were treated without or with2-bromopalmitate for 3 hours, then either were left unactivated (−) oractivated (+) with OKT3 mAb (0.3 mg/ml) for 3 minutes and lysed. Lysateswere subjected to SDS-PAGE followed by immunoblotting with antiphosphotyrosine antibody (PY99). FIG. 5B: cells were treated and lysedas described in (FIG. 5A). Lysates were immunoprecipitated withagarose-conjugated phophotyrosine antibody (PY99) and immunblotted forspecific proteins. Alternatively, lysates were immunoprecipitated forspecific proteins and immunoblotted with antiphosphotyrosine antibody,as depicted in the figure.

FIGS. 6A-1 and 2 shows calcium mobilization in Jurkat T cells. FIG. 6A:untreated cells were preincubated with Fluo 3 as indicated below andcalcium release was measured by flow cytometry. Top: After obtaining abackground fluorescence (Baseline), cells were activated with OKT3 mAb,and fluorescence was measured for the indicated time (post OKT3).Bottom: Quantations of fluorescence before (left—baseline) and after(right—post OKT3) CD3 stimulation.

FIGS. 6B-1 and 2 cells were pretreated with 2-bromopalmitate andanalyzed as described in FIGS. A-1 and 2).

FIG. 7 shows the activation of MAP Kinase in Jurkat Cells. Cells weretreated with 2-bromopalmitate, activated with OKT3 mAb and lysed.Lysates were subjected to SDS-PAGE and immublotted for active(Top—pERK), or for total (bottom—MAPK) MAP Kinase.

FIG. 8 shows the effect of polyunsaturated fatty acids on Fyn fattyacylation and localization to detergent resistant microdomains in COS-1cells. Cells expressing Fyn were preincubated overnight with 50 μMarachidonic acid (20:4) or eicosapentaenoic acid (20:5) or leftuntreated (C), as described below. FIG. 8A shows the cells wereradiolabeled for 4 hours in the absence (C) or presence of 20:4 or 20:5with I¹²⁵IC13 or I¹²⁵-IC16, lysed and duplicate samples were subjectedto immunoprecipitation with anti-Fyn antibody. Lysates were subjected toSDS-PAGE and phosphorimaging (top). Bottom: to monitor total proteinlevels, aliquots from each sample were subjected to SDS-PAGE followed byimmunoblotting with anti-Fyn antibody. FIG. 8B and FIG. 8C: Quantitationof (FIG. 5A). Effect of polyunsaturated fatty acids on IC13 (FIG. 8B) orIC16 (FIG. 8C) incorporation into Fyn. Bars represent the average of 3sets of duplicate experiments. FIG. 8D: DRM localization. Cells werelysed with buffer containing 0.5% Triton X-100, and layered on thebottom of a sucrose gradient as detailed below. After overnightcentrifugations, 1 ml fractions were collected and the DRM localizationof Fyn was analyzed by SDS-PAGE and immunoblotting with anti-Fynantibody.

DETAILED DESCRIPTION OF THE INVENTION

This invention describes a palmitate analog, 2bromopalmitate, thateffectively blocks Fyn fatty acylation in general, and palmitoylation inparticular. Treatment of COS-1 cells with 2-bromopalmitate blockedmyristoylation and palmitoylation of Fyn, and inhibited membrane bindingand localization of Fyn to detergent resistant membranes (DRMs)¹. InJurkat T cells, 2-bromopalmitate blocked localization of the endogenouspalmitoylated proteins Fyn, Lck and LAT to detergent resistantmicrodomains. This resulted in impaired signaling through the T cellreceptor as evidenced by reductions in tyrosine phosphorylation, calciumrelease and activation of MAP kinase. The polyunsaturated fatty acidsarachidonic acid and eicosapentaenoic acid inhibit Fyn palmitoylationand consequently block Fyn localization to detergent resistantmicrodomains.

¹Abbreviations: DRMs, detergent resistant microdomains, ITAM,immune-receptor tyrosine-based activation motif; PUFAs, polyunsaturatedfatty acids; GFP, Green Fluorescent Protein; IC13, 13-iodotridecanoicacid; IC16, 16-iodohexadecanoic acid; NMT, N-myristoyl transferase; PAT,palmitoyl acyl transferase; TCR, T cell receptor.

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Maniatis, Fritsch & Sambrook,“Molecular Cloning: A Laboratory Manual (1982); “DNA Cloning: APractical Approach,” Volumes I and II (D. N. Glover ed. 1985);“Oligonucleotide Synthesis” (M. J. Gait ed. 1984); “Nucleic AcidHybridization” [B. D. Hames & S. J. Higgins eds. (1985)]; “Transcriptionand Translation” [B. D. Hames & S. J. Higgins eds. (1984)]; “Animal CellCulture” [R. I. Freshney, ed. (1986)]; “Immobilized Cells And Enzymes”:[IRL Press, (1986)]; B. Perbal, “A Practical Guide To Molecular Cloning”(1984).

The present invention is directed to a method of inhibiting Fyn/Lckfatty acylation and protein palmitoylation in a cell in an individual inneed of such treatment comprising the step of administering to saidindividual a pharmacologically effective dose of 2-bromopalmitate.Preferably, the 2-bromopalmitate is administered in a dose of from about0.1 mg/kg to about 100 mg/kg of total body weight of said individual.Administration of 2-bromopalmitate inhibits N-terminally palmitoylatedproteins, myristoylation of proteins and T cell signalling events. Inone aspect, the individual has a autoimmune disease. Representativeexamples of autoimmune disease include rheumatoid arthritis, Crohn'sdisease, diabetes, multiple sclerosis and systemic lupus erythematosus.

The present invention is directed to a method of treating an individualhaving a pathophysiological state comprising the step of administeringto said individual a pharmacologically effective dose of2-bromopalmitate. Preferably, the individual has an autoimmune diseaseor abnormal T cell signalling.

The present invention is also directed to a pharmaceutical compositionscontaining 2-bromopalmitate. In such a case, the pharmaceuticalcomposition comprises 2-bromopalmitate and a pharmaceutically acceptablecarrier. A person having ordinary skill in this art would readily beable to determine, without undue experimentation, the appropriatedosages and routes of administration of 2-bromopalmitate.

Compounds of the present invention, pharmaceutically acceptable saltthereof and pharmaceutical compositions incorporating such, may beconveniently administered by any of the routes conventionally used fordrug administration, e.g., orally, topically, parenterally, or byinhalation. 2-bromopalmitate may be administered in conventional dosageforms prepared by combining the compound with standard pharmaceuticalcarriers according to conventional procedures. 2-bromopalmitate may alsobe administered in conventional dosages in combination with a known,second therapeutically active compound. These procedures may involvemixing, granulating and compressing or dissolving the ingredients asappropriate to the desired preparation. It will be appreciated that theform and character of the pharmaceutically acceptable carrier or diluentis dictated by the amount of active ingredient with which it is to becombined, the route of administration and other well known variable. Thecarrier(s) must be “acceptable” in the sense of being compatible withthe other ingredients of the formulation and not deleterious to therecipient thereof.

The pharaceutical carrier employed may be, for example, either a solidor a liquid. Representative solid carriers are lactose, terra alba,sucrose, talc, gelatin, agar, pectin, acacia, magnesium sterate, stearicacid and the like. Representative liquid carriers include syrup, peanutoil, olive oil, water and the like. Similarly, the carrier may includetime delay material well known in the art such as glyceryl monosterateor glyceryl disterarate alone or with a wax.

A wide variety of pharmaceutical forms can be employed. Thus, if a solidcarrier is used, the preparation can be tableted, placed in a hardgelatin capsule in powder or pellet form or in the form of a troche orlozenge. The amount of solid carrier will vary widely but preferablywill be from about 25 mg to about 1 gram. When a liquid carrier is used,the preparation will be in the form of a syrup, emulsion, soft gelatincapsule, sterile injectable liquid such as an ampule or nonaqueousliquid suspension.

2-bromopalmitate may be administered topically (non-systemically). Thisincludes the application of 2-bromopalmitate externally to the epidermisor the buccal cavity and the instillation of such a compound into theear, eye and nose, such that the compound does not significantly enterthe bloodstream. Formulation suitable for topical administration includeliquid or semi-liquid preparations suitable for penetration through theskin to the site of inflammation such as liniments, lotions, creams,ointments, pastes and drops suitable for administration to the ear, eyeand nose. The active ingredient may comprise, for topical administrationfrom 0.001% to 10% w/w, for instance from 1% to 2% by weight of theFormulation. It may however, comprise as much as 10% w/w but preferablywill comprise less than 5% w/w, more preferably from 0.1% to 1% w/w ofthe Formulation.

Lotions according to the present invention include those suitable forapplication to the skin and eye. An eye lotion may comprise a sterileaqueous solution optionally containing a bactericide and may be preparedby methods similar to those for the preparation of drops. Lotions orliniments for application to the skin may include an agent to hastendrying and to cool the skin, such as an alcohol or acetone, and/or amoisterizer such as glycerol or an oil such as castor oil or arachisoil.

Creams, ointments or pastes according to the present invention aresemi-solid formulations of the active ingredient for externalapplication. They may be made by mixing the active ingredient in finelydivided or powdered form, alone or in solution or suspension in anaqueous or non-aqueous fluid, with the aid of suitable machinery, with agreasy or non-greasy base. The base may comprise hydrocarbons such ashard, soft or liquid paraffin, glycerol, beeswax, a metallic soap, amucilage, an oil of natural origin such as almond, corn, archis, castor,or olive oil; wool fat or its derivatives or a fatty acid such as stericor oleic acid together with an alcohol such as propylene glycol or amacrogel. The formulation may incorporate any suitable surface activeagent such as an anionic, cationic or non-ionic ionic surfactant such asa sorbitan ester or a polyoxyethylene derivative thereof. Suspendingagents such as natural gums, cellulose derivatives or inorganicmaterials such as silicaceous silicas, and other ingredients such aslanolin may also be included.

Drops according to the present invention may comprise sterile aqueous oroily solutions or suspensions and may be prepared by dissolving theactive ingredient in a suitable aqueous solution of a bactericidaland/or fungicidal agent and/or any other suitable preservative, andpreferably including a surface active agent. The resulting solution maythen be clarified by filtration, transferred to a suitable containerwhich is then sealed and sterilized by autoclaving. Alternatively, thesolution may be sterilized by filtration and transferred to thecontainer by an aseptic technique. Examples of bactericidal andfungicidal agents suitable for inclusion in the drops are phenymercuricnitrate or acetate (˜0.002%), benzalkonium chloride (˜0.01%) andchlorhexidine acetate (˜0.01%). Suitable solvents for the preparation ofan oily solution include glycerol, diluted alcohol and propylene glycol.

2-bromopalmitate may be administered parenterally, i.e., by intravenous,intramuscular, subcutaneous, intranasal, intrarectal, intravaginal orintraperitoneal administration. The subcutaneous and intramuscular formsof parenteral administration are generally preferred. Appropriate dosageforms for such administration may be prepared by conventionaltechniques. Compounds may also be administered by inhalation, e.g.,intranasal and oral inhalation administration. Appropriate dosage formsfor such administration, such as aerosol formulation or a metered doseinhaler may be prepared by conventional techniques well known to thosehaving ordinary skill in this art.

For all methods of use disclosed herein for 2-bromopalmitate, the dailyoral dosage regiment will preferably be from about 0.1 to about 100mg/kg of total body weight. The daily parenteral dosage regimen willpreferably be from about 0.1 to about 100 mg/kg of total body weight.The daily topical dosage regimen will preferably be from about 0.01 toabout 1 g, administered one to four, preferably two to three timesdaily. It will also be recognized by one of skill in this art that theoptimal quantity and spacing of individual dosages of 2-bromopalmitate,or a pharmaceutically acceptable salt thereof, will be determined by thenature and extent of the condition being treated and that such optimumscan be determined by conventional techniques.

Suitable pharmaceutically acceptable salts are well known to thoseskilled in the art and include basic salts of inorganic and organicacids, such as hydrochloric acid, hydrobromic acid, sulphuric acid,phophoric acid, methane sulphonic acid, ethane sulphonic acid, aceticacid, malic acid, tartaric acid, citric acid, lactic acid, oxalic acid,succinic acid, fumaric acid, maleic acid, benzoic acid, salicylic acid,phenylacetic acid and mandelic acid. In addition, pharmaceuticallyacceptable salts of 2-bromopalmitate may also be formed with apharmaceutically acceptable cation, for instance, if a substituent groupcomprises a carboxy moiety. Suitable pharmaceutically acceptable cationsare well known in the art and include alkaline, alkaline earth ammoniumand quaternary ammonium cations.

Autoimmune diseases are characterized by immune cell destruction of selfcells, tissues and organs. Representative examples of such autoimmunediaseases are rheumatoid arthritis diabetes, multiple sclerosis, Crohn'sdisease and systemic lupus erythematosus. 2-bromopalmitate has potentialuses in other immune cell functions. For example, the IgE receptor usespalmitoylated Lyn (another Src kinase family member) to signal for theinflammatory response and Lyn must be palmitoylated and in membranerafts in order to function. Thus, 2-bromopalmitate could be used as ananti-inflammatory agent.

The following examples are given for the purpose of illustrating variousembodiments of the invention and are not meant to limit the presentinvention in any fashion.

EXAMPLE 1

Cell Culture and Transfections

COS-1 cells were maintained and transfected as previously described (9).Transfection with FUGENE™ 6 Transfection Reagent (Boehringer Mannheim)was carried out according to the manufacturer's instructions. Jurkat Tcells were maintained in RPMI 1640 supplemented with 10% FBS, 100 μg ofpenicillin and streptomycin per ml and 100 μg of sodium pyruvate andglutamine per ml. Cells were transfected by electroporation aspreviously described (38).

EXAMPLE 2

Antibodies

Monoclonal anti-Fyn and anti-Lck antibodies used for Western blottingwere purchased from Transduction Laboratories (Lexington, Ky.). Therabbit polyclonal antiserum to Fyn used for immunoprecipitation wasdescribed previously (13). Monoclonal anti-PLCγ-1 and rabbit polyclonalsanti-LAT, anti PI3 kinase, anti-Vav and anti-ZAP-70 were purchased fromUpstate Biotechnology (Lake Placid, N.Y.). Monoclonals anti-Hras,anti-p-ERK anti-P-Tyr (PY99) and agarose-conjugated PY99 were purchasedfrom Santa Cruz Biotechnology (Santa Cruz, Calif.). Anti MAPK antibodywas purchased from New England Biolabs (Beverly, Mass.). Fluorescein(FITC)-conjugated Goat Anti-Mouse secondary antibody was purchased fromJackson ImmunoResearch Laboratories (West Grove, Pa.). Anti GFP antibodywas purchased from CLONTECH Laboratories (Palo Alto, Calif.).

EXAMPLE 3

Fyn Chimeras

The Fyn Chimeras Gα_(o)(10)Fyn and GAP43(10)-Fyn have been describedpreviously (12). G2A, C35 Fyn-HRas was constructed as follows. Anantisense oligonucleotide primer was designed that corresponded to thelast 6 amino acids of Fyn fused in frame to the C-terminal 12 aminoacids of H-Ras, followed by a stop codon, a SalI site and a GIC clamp. Asense primer that began 57 bases upstream of a unique BglII site in Fynwas constructed. These two primers were used in a PCR reaction toamplify a fragment containing the C-terminal region of Fyn fused to theH-Ras tail. The PCR reaction product was cut with BglII and SalI andused to replace the corresponding region of Fyn in G2AFyn/pSP65. G2A FynHRas/pSP65 was then digested with NcoI and BglII to remove the 5′ codingregion of Fyn, and ligated to a 1.7 kb NcoI/BglII fragment from anotherFyn clone containing the G2A, C3S mutation. G2A, C3S Fyn-HRas/pSP65 wasdigested with EcoRI and SalI and ligated into EcoRI and SalI cut pCMV5.The construct was verified by DNA sequencing prior to use intransfections.

EXAMPLE 4

Cell Labeling

The syntheses of 13-[¹²⁵I]-iodotridecanoic acid (IC13)or16-[¹²⁵I]-iodohexanoic acid (IC16) were carried out as describedpreviously (39). Cell labeling was carried out as described (12,13) withmodifications. Briefly, each 60 mm plate of Fyn transfected COS-1 cellswas incubated O/N in DMEM containing 2.5% FBS, 0.5% defatted BSA (Sigma)with or without 100 μM 2-bromopalmitate or 50 μM polyunsaturated fattyacid. Prior to labeling, the cells were incubated for 1 hr with 1 ml ofDMEM containing 2% dialyzed FBS, then labeled for 4 hours with 25-50 μCiIC13 or IC16 in DMEM containing 2% dialyzed FBS, 0.5% defatted BSA withor without 2-bromopalmitate or polyunsaturated fatty acid. Labeled cellswere washed three times with cold STE (100 mM NaCl, 10 mM Tris pH 7.4, 1mM EDTA) and lysed in 0.6 ml of cold RIPA buffer containing proteaseinhibitors (10 μg/ml each of benzamidine, AEBSF, TPCK and TLCK, 1.5μg/ml each of Leupeptin, Pepstatin A and Aprotinin). Lysates wereclarified at 100,000×g for 15 min at 4° C. in a Beckman TL-100ultracentrifuge. Lysates were immunoprecipitated with rabbit anti-Fynantibody and protein A-agarose. Immunoprecipitates were washed threetimes with cold RIPA buffer and suspended in 1× sample buffer containing100 mM dithiothreitol and subjected to SDS-PAGE. Gels were dried betweencellophane and analyzed by phosphorimaging after 12-36 hours exposure.Experiments in Jurkat T cells were carried out according to the aboveprotocol using 2×10⁷ cells per experiment, and reducing the incubationtime with 2-bromopalmitate to 3-4 hours.

EXAMPLE 5

Subcellular Cell Fractionation

Each 60 mm plate of Fyn transfected COS-1 cells was starved for 1 hourin DMEM containing 2.5% FBS and 0.5% defatted BSA with or without 100 μM2-bromopalmitate. After overnight culture at 37° C., the cells werefractionated into P100 and S100 fractions, immunoprecipitated withanti-Fyn antibody, subjected to SDS-PAGE, and immunoblotted withanti-Fyn antibody as previously described (9,12,13).

Analysis of the G2A,C3S Fyn-HRas chimera was performed according to theprocedure described above. Analysis of the G12V HRas construct wasaccording to the procedure described above, except that the samples werenot immunoprecipiated, and were subjected to immunoblotting withanti-HRas antibody.

For analysis of newly synthesized Fyn, cells were cultured overnight asdescibed above, then starved for 1 hr in DMEM minus methionine andcysteine containing 2% dialyzed FBS, 0.5% defatted BSA with or without100 μM 2-bromopalmitate. Cells were labeled for 5 minutes withTrans³⁵S-Label (ICN, Irvine, Calif.), then fractionated as describedabove. Gels were treated for 20 minutes with 1M salicylic acid prior todrying. The Gαo(10)-Fyn chimera was labeled for 5 min and GAP-43 Fynchimera was labeled for 2 hours and fractionated according to the aboveconditions.

EXAMPLE 6

Immunofluorescence Microscopy

COS-1 cells were transfected with a Fyn(16)-eGFP construct (12) andseeded onto 25-mm glass coverslips 2 days prior to the experiment. Cellswere treated overnight with or without 2 bromopalmitate as describedabove. Plates were washed with PBS and coverslips were mounted ontoglass slides in PBS and observed with a 40× and 100×oil immersion lenson a Zeiss Axiophot 2 microscope and photographed with Kodak TMAX 400.

EXAMPLE 7

T cell Activation and Phosphotyrosine Immunoblots

Jurkat T cells (2×10⁶−1×10⁷) were centrifuged at 1,000×g for 5 minutes,rinsed with RPMI, resuspended in RPMI supplemented with 2% dialyzed FBS,0.5% defatted BSA, and incubated with or without 100 μM 2-bromopalmitateat 8×10⁵ cells/ml for 3 hours. The cells were centrifuged, washed withRPMI and resuspended in RPMI at 1×10⁷−1×10⁸ cells/ml. The cells werethen activated with anti-CD3 OKT3 mAb (0.3 mg/ml) for 3 min at 37° C.,quickly spun down, washed once with cold RPMI and once with cold STE,and lysed in RIPA Samples were solubilized in 1× sample buffercontaining 5% β-mercaptoethanol and subjected to SDS-PAGE, followed byimmunoblotting with anti-phosphotyrosine antibody (PY99). For analysisof specific proteins, RIPA lysates were immunoprecipitated with thespecific antibodies O/N, and immunoblotted for phosphotyrosine.Alternatively, proteins were immunoprecipitated with agarose-conjugatedanti phosphotyrosine antibody and blotted for the specific proteins.

EXAMPLE 8

Isolation of DRMs

Isolation of Triton X-100 resistant and soluble fractions was carriedout as described previously (12). Isolation of detergent resistantmicrodomains by sucrose gradients were carried out as follows (19):Jurkat cells (5×10⁷) were treated with 2-bromopalmitate, activated withOKT3 mAb as described above, and lysed in 1 ml lysis buffer (25 mM MESpH 6.5, 150 mM NaCl, 0.5% Triton X-100, 1 mM Na₃VO₄) supplemented withprotease inhibitors for 30 minutes at 0° C. After homogenizing 10 timeswith a loose fit Dounce homogenizer, lysates were mixed with 1 ml 85%sucrose in MBS (25 mM MES pH 6.5, 150 mM NaCl), and overlayered with 6ml 30% sucrose in MBS, then with 4 ml 5% sucrose in MBS. Followingcentrifugation for 16 hours at 145,000 g in an SW40 rotor, 1 mlfractions were collected and analyzed by PAGE and immunoblotting withanti-Fyn, anti-LAT, or anti-Lck antibodies.

For isolation of detergent resistant microdomains in PUFA-treated COS-1cells, a confluent 100 mm plate of COS-1 cells transiently transfectedwith Fyn cDNA was washed with S-IE and subjected to the same proceduredescribed above. Fractions were analyzed for the presence of Fyn byimmunoblotting.

EXAMPLE 9

Calcium Mobilization Assay

Jurkat cells were treated with 2-bromopalmitate as described above. Thecells (2×10⁵) were collected by centrifugation and resuspended at 2×10⁶cells/ml in RPMI containing in 50 μM fluo-3 with or without 100 μM2-bromopalmitate for 30 minutes at room temperature. Cells loaded withfluo-3 were then collected by centrigation, washed with Hank's BufferedSaline Solution (5.4 mM KCl, 0.3 mM Na₂HPO₄, 0.4 mM KH₂PO₄, 4 mM NaHCO₃,1.3 mM CaCl₂, 0.5 mM MgCl₂, 0.6 mM MgSO₄, 137 mM NaCl, 5.6 mM glucose,20 mM Hepes pH 7.4) and resuspended in the same buffer at 5×10⁵ cells/mlat 37° C. To initiate calcium flux, the cells were activated with OKT3antibody as described above, and analyzed for free calcium ion bymeasurement of fluo-3 fluoresence emission by flow cytometry.

For analysis of CD3 positive cells, 1×10⁶ cells were centrifuged, washedand resuspended in 100 μl ice cold phosphate buffered saline (PBS-136 mMNaCl, 2.6 mM KCl, 4.3 mM Na₂HPO₄, 1.5 mM KH₂PO₄) containing 1% FBS. OKT3antibody was added to a final concentration of 0.3 mg/ml. Following 30minutes on ice, the cells were washed twice with PBS/1% FBS andincubated at 0° C. for an additional 30 minutes with an FITC-conjugatedGoat Anti-Mouse secondary antibody (1:20 dilution). The cells werewashed twice, resuspended in PBS/1% FBS, and subjected to FACS analysis.

EXAMPLE 10

Activation of MAP Kinase

Jurkat cells were treated with 2-bromopalmitate and activated asdescribed above. Lysates were analyzed for the presence of active MAPkinase by immunoblotting with a pERK antibody, and for total MAP kinaseby immunoblotting with an anti MAPK antibody.

EXAMPLE 11

Identification of 2-Bromopalmitate as an Inhibitor of Fyn FattyAcylation

A number of palmitic acid analogs were screened for their ability toinhibit Fyn palmitoylation. COS-1 cells were transfected with cDNAencoding Fyn. Three days after transfection, the cells were labeled witheither [³⁵S]-methionine, 13-[¹²⁵I]-iodotridecanoic acid (IC13), aniodinated myristate analog, or 16-[¹²⁵I]-iodohexanoic acid (IC16), aniodinated palmitic acid analog (39), in the presence or absence ofnonradioactive palmitate analogs. Cells were lysed, immunoprecipitatedwith anti-Fyn antibody, and analyzed by SDS-PAGE and phosphorimaging.2-Bromo-palmitate efficiently inhibited Fyn fatty acylation (FIG. 1A).When normalized for total protein levels, 70% of Fyn myristoylation andover 90% of Fyn palmitoylation was inhibited in the presence of2-bromopalmitate. Treatment of cells with other analogs, including2-hydroxypalmitate, palmitoleic acid and 16-hydroxypalmitate had noeffect (data not shown).

EXAMPLE 12

Effect of 2-bromo-palmitate on Subcellular Localization of Fyn

Newly synthesized Fyn becomes plasma membrane bound within 5 minutesafter biosynthesis (12). The rapid membrane targeting is dependent ondual fatty acylation of Fyn with myristate and palmitate. The effect of2-bromopalmitate on the ability of newly synthesized Fyn to localize tomembranes was next examined. Transfected COS-1 cells were incubated for12-16 hours with or without 100 μM 2-bromopalmitate. Cells were thenmetabolically labeled with [³⁵S]-methionine for 5 minutes followed byfractionation into cytosolic (S100) or membrane (P100) fractions.

As depicted in FIG. 1B, in untreated cells, 90% of the labeled Fyn wasmembrane bound. In cells treated with 2-bromopalmitate, 50% of Fynremained cytosolic, demonstrating the ability of the reagent topartially block membrane association of newly synthesized Fyn. Theeffect of 2-bromo-palmitate on membrane localization of steady-state Fynwas also examined. Transfected cells were treated with 2-bromo-palmitateas described above, then fractionated, immunoprecipitated with anti-Fynfollowed by Western blotting with anti-Fyn antibody. The effect of2-bromo-palmitate on membrane localization of steady-state Fyn wasidentical to the effect on newly synthesized Fyn, with 50% of the Fynprotein fractionating in the cytosol (data not shown). These resultsmimic the fractionation pattern of a non-palmitoylated Fyn mutant(C3,6SFyn), and of a non-myristoylated Fyn mutant (G2AFyn), and stronglysuggest that the redistribution of Fyn observed in 2-bromopalmitatetreated cells is due to inhibition of Fyn fatty acylation (13).

Previous experiments have shown that following rapid membrane binding ofnewly synthesized Fyn, there is a slower partitioning of Fyn (10-20minutes) to regions of the plasma membrane that are resistant to TritonX-100 extraction at 4° C. (12). Therefore the effect of 2-bromopalmitateon the localization of Fyn to Triton X-100 insoluble fractions wasexamined. Transfected COS-1 cells were left untreated or treated with2-bromopalmitate as described above. Cells were extracted with buffercontaining 1% Triton X-100, and samples were subjected toimmunoprecipitation and Western blotting with anti-Fyn antibodies.

As depicted in FIG. 1C, in untreated cells the majority of Fyn wasassociated with detergent resistant fractions (R), in agreement withprevious experiments (12). In comparison, Fyn in 2-bromopalmitatetreated cells was mostly soluble, demonstrating the ability of2-bromopalmitate to partially block association of Fyn with detergentresistant membrane subdomains.

To investigate the effect of 2-bromopalmitate on the intracellularlocalization of Fyn more precisely, COS-1 cells expressing aFyn(16)-eGFP construct were examined by immunofluorescence. Thisconstruct contains the first 16 amino acids of Fyn fused in frame toeGFP; the chimera is targeted to the plasma membrane and detergentresistant microdomains (12). Cells were cultured with no treatment, with2-bromopalmitate or with 2-hydroxymyristate, a known myristoylationinhibitor (40,41), as described above, and were examined live byfluorescence microscopy.

FIG. 2A shows that Fyn(16)-eGFP is primarily distributed in the plasmamembrane (Top). In contrast, cells treated with 2-bromopalmitate(middle) and 2-hydroxymyristate (bottom) showed reduced plasma membranestaining, and instead exhibited a distinct perinuclear staining,presumably representing cytosolic and intracellular membranedistribution. Analysis of the subcellular localization of Fyn(16)-eGFPin the presence of 2-bromopalmitate and 2-hydroxymyristate was performedas described above for steady-state Fyn. As depicted in FIG. 2B, in theabsence of treatment, 80% of Fyn(16)-eGFP Fyn was membrane bound,whereas in cells treated with 2-bromopalmitate and 2-hydroxymyristate,65% and 78% of Fyn(16)-eGFP were cytosolic, respectively. These resultsstrengthen the hypothesis that fatty acylation of Fyn is important forthe proper localization of the protein within the cell.

EXAMPLE 13

Effect of 2-bromo-palmitate on Membrane Localization of OtherPalmitoylated Proteins

Palmitoylation has been shown to occur on a wide variety of cellularproteins and the sites of palmitoylation can be quite diverse. Whether2-bromopalmitate can inhibit palmitoylation and membrane binding ofother palmitoylated proteins was next examined. Three representativepalmitoylated protein sequences were chosen as model systems. The Gαosubunit of the heterotrimeric Go protein is myristoylated andpalmitoylated on an N-terminal Gly-Cys motif, similar to Fyn and Lck(42,43). The neuronal protein GAP43 (neuromodulin) is palmitoylated nearthe N-terminus at cysteines 3 and 4, but is not myristoylated (44).Finally, the oncogenic H-Ras protein is palmitoylated just upstream ofthe C-terminal CAAX box (45).

Each of these three sequences was appended onto the Fyn protein.Gαo(10)-Fyn and GAP43(10)-Fyn are chimeric constructs with the first 10amino acids of Gαo or GAP-43, respectively, in place of the first 10amino acids of wt Fyn. These constructs have been previously described(12,13). In addition, the H-Ras tail was fused to the C-terminus of anon-acylated Fyn mutant (G2A,C3SFyn-HRas). This construct contains fulllength Fyn with mutations in the N-terminal myristoylation andpalmitoylation sites, but with the C-terminus of H-Ras available forprenylation and palmitoylation.

Finally, an oncogenic G12V full length H-Ras construct was tested. Asdepicted in FIGS. 3A and B, 2-bromopalmitate inhibited membrane bindingof the two N-terminal palmitoylated proteins, GαO(10)-Fyn andGAP43(10)-Fyn, to the same extent as wt Fyn. In contrast,2-bromopalmitate had only a minimal effect on the membrane localizationof the two Ras constructs, inducing a 10-20% shift from membrane tocytosol (FIGS. 3C and D). The G12V H-Ras construct migrates as a doubleton a gel. The slower migrating form represents the non-processedcytosolic form of H-Ras, and the faster migrating form represents theprocessed Ras, which is membrane bound. Thus 2-bromopalmitate appears topossess some specificity towards inhibiting membrane localization ofN-terminal palmitoylated proteins, in comparison to a proteinpalmitoylated near the C-terminus. The absolute sequence surrounding thepalmitoylated cysteine residue did not seem to be important, as theGAP43(10)-Fyn construct was affected to the same extent as wt Fyn andGαo(10)-Fyn.

EXAMPLE 14

2-bromopalmitate Inhibits Fatty Acylation and Localization ofPalmitoylated Proteins to DRMs in T Cells

The Src family kinases Fyn and Lck play critical roles in T cellreceptor (TCR) mediated signaling. Palmitoylation of Fyn and Lck hasbeen shown to be essential for localization to detergent resistantmicrodomains in T cells, and localization to detergent resistantmicrodomains is required for efficient signaling by the activated TCR(11). The ability of 2-bromopalmitate to inhibit Fyn fatty acylation wastested in Jurkat T cells. Cells were transfected by electroporation withcDNA encoding Fyn. Two days after transfection, cells were labeled withIC13 or IC16 as described above for COS cells. Total protein levels weremonitored by immunoprecipitation followed by immunoblotting withanti-Fyn antibody.

As depicted in FIG. 4A, myristoylation and palmitoylation were inhibitedby 75% and 90% respectively in 2-bromopalmitate treated cells, relativeto untreated controls, demonstrating the ability of the reagent toinhibit Fyn fatty acylation in Jurkat T-cells.

The ability of 2-bromopalmitate to inhibit localization of palmitoylatedproteins to detergent resistant microdomains in activated Jurkat cellswas examined next. Cells were either left untreated or treated with 100μM 2-bromopalmitate for 3 hours, washed, resuspended in serum-freemedia, and the TCR was activated with OKT3 mAb. Activated cells wereextracted with Triton X-100 containing buffer. Lysates were layered onthe bottom of a sucrose gradient as described above, and subjected toovernight ultracentrifugation. Rafts, which contain detergent resistantmicrodomains, were collected at the 35%/5% sucrose interface (FIG. 4B,fractions 8-11), whereas fractions 1-4 represented the Triton solublefractions (FIG. 4B). Each fraction was analyzed by immunoblotting withspecific antibodies. 59% of Lck was found in the rafts in control cells,compared with 19% in cells treated with 2-bromopalmitate (Top).Likewise, the amount of Fyn found in the rafts was 88% in control cells,but only 59% in treated cells (middle).

The effect of 2-bromopalmitate on localization of LAT, anotherpalmitoylated protein in T cells that has been shown to be localized toplasma membrane rafts (10,20) was also examined. The majority of LAT(71%) was found in the detergent resistant microdomains in controlcells, whereas in cells treated with 2-bromopalmitate only 41% of theprotein remained associated with this fraction (bottom). These dataindicate that in Jurkat T-cells, 2-bromopalmitate is able to partiallyblock association of endogenous Fyn, Lck and LAT with rafts.

EXAMPLE 15

Effect of 2-bromopalmitate on Tyrosine Phosphorylation in T Cells.

One of the earliest signaling events after T cell receptor activation isthe tyrosine phosphorylation of multiple intracellular proteins. Theinitial phosphorylation events are mediated by activation of Src familykinases. Whether 2-bromopalmitate can interfere with signaling throughthe T cell receptor was examined by analyzing the ability of thecompound to block tyrosine phosphorylation in activated T cells.

Jurkat cells were incubated with 2-bromopalmitate and activated withOKT3 anti-CD3 antibody. Cell lysates were analyzed by immunoblottingwith anti phosphotyrosine antibodies. In control cells, stimulation withOKT3 antibodies induced tyrosine phosphorylation of multiple proteins(Fig 5A). In cells treated with 2-bromopalmitate, the phosphorylation ofseveral proteins was significantly inhibited. The most dramatic effectwas on a 36 kDa protein, which represents LAT (see below).

In order to identify the individual proteins whose tyrosinephosphorylation is affected by 2-bromopalmitate, lysates wereimmunoprecipitated with a panel of specific antibodies, andimmunoblotted for phosphotyrosine. Alternatively, lysates wereimmunoprecipitated with an antiphosphotyrosine antibody, and blottedwith antibodies to specific proteins.

FIG. 5B shows that in 2-bromopalmitate treated cells, CD3-mediatedtyrosine phosphorylation of LAT was inhibited completely. PLC-γlphosphorylation was inhibited by 70%, Vav phosphorylation was inhibitedby 40%, ZAP-70 phosphorylation was inhibited by 50%, and PI3Kphosphorylation was inhibited by 50%. Low to moderate increases intyrosine phosphorylation were observed in the presence of2-bromopalmitate alone for some of the proteins. The reason for thisbasal activation is unknown.

To verify that the observed inhibition of T cell receptor-mediatedtyrosine phosphorylation was not a result of toxicity effects of2-bromopalmitate, aliquots of each sample were analyzed byimmunoblotting with anti-LAT and anti-actin antibodies. The levels ofLAT and actin were not affected by 2-bromopalmitate (data not shown).Thus 2-bromopalmitate was able to inhibit signaling through the T cellreceptor, as assayed by its ability to inhibit tyrosine phosphorylationof key substrate proteins.

EXAMPLE 16

2-bromopalmitate Inhibits Calcium Mobilization in T Cells

T cell receptor activation results in increased Ca⁺⁺ mobilization instimulated T cells. The increase in Ca⁺⁺ flux is mediated by tyrosinephosphorylation and activation of PLC-γl. PLC-γl hydrolyzesphosphatidylinositol 4,5-bisphosphate (PIP2) to inositol1,4,5-triphosphate (IP3), which promotes calcium release from the ER(46). The ability of 2-bromopalmitate to interfere with calcium releasewas assayed next by flow cytometry.

Jurkat cells were incubated with or without 2-bromopalmitate, washed andstained with the fluorescent dye fluo-3 (50 μM). Cells were activatedwith OKT3 antibody and analyzed by flow cytometry. FIG. 5 shows that inresponse to T cell receptor activation, T cells treated with2-bromopalmitate were severely impaired in their ability to releasecalcium compared with control cells (FIGS. 6A and 6B). Quantitation ofthe data revealed that calcium flux shut down almost completely in thepresence of 2-bromopalmitate. No effect of 2-bromopalmitate on cellsincubated in the absence of OKT3 was noted.

To ensure that the observed inhibition of calcium flux was not due to adecreased expression of CD3 in 2bromo-palmitate treated cells, Jurkatcells were incubated with OKT3 antibody at 0° C., followed by incubationwith a Fluorescein (FITC) conjugated Goat Anti-Mouse secondary antibody.The percentage of CD3 positive cells was analyzed by FACS analysis. Over95% of the cells were found to be positive for CD3 in control and2-bromopalmitate treated cells (data not shown).

EXAMPLE 17

2-bromopalmitate Inhibits MAP Kinase Activation

One of the proximal events following T cell receptor engagement isactivation of the MAP Kinase pathway. The ability of 2-bromopalmitate toinhibit activation of MAP kinase was examined in Jurkat cells. Cellswere cultured with or without 2bromo-palmitate and activated asdescribed above. Cell lysates were subjected to SDS-PAGE andimmunoblotted with anti-active MAPK kinase (pERK1). As depicted in FIG.7, 2-bromopalmitate inhibited the activation of MAPK kinase by 70%. Thelevels of total MAPK kinase remained unchanged (FIG. 7).

EXAMPLE 18

PUFAs Inhibit Fyn Palmitoylation and Localization to DRMs in COS-1 Cells

The data reported above identify 2-bromopalmitate as an inhibitor ofprotein fatty acylation and T cell receptor-mediated signaling. Whetherother fatty acids, particularly long chain unsaturated compounds, mightalso interfere with protein fatty acylation was examined next. It hasrecently been reported that polyunsaturated fatty acids inhibit T cellsignal transduction by displacing Src kinases Fyn and Lck from thedetergent resistant microdomains (37). This inhibition was speculated tobe due to polyunsaturated fatty acid-induced disruption of DRM structureand composition. Based on these results with 2-bromopalmitate,polyunsaturated fatty acid-induced displacement of Fyn/Lck from thedetergent resistant microdomains may actually be due to alterations ofS-acylation.

To test this hypothesis, Fyn transfected COS-1 cells were incubated O/Nwith or without 50 μM arachidonic acid (20:4) or eicosapentaenoic acid(20:5), then labeled with IC13 or IC16. Total protein levels weremonitored by immunoblotting aliquots of each sample with anti-Fynantibody (FIG. 8A, lower panel).

As depicted in FIG. 8A, Fyn myristoylation was not affected bypolyunsaturated fatty acid treatment to a significant effect (FIG. 8B).On the other hand, Fyn palmitoylation was affected quite dramatically.Arachidonic acid inhibited incorporation of IC16 into Fyn by 55%, andeicosapentaenoic acid by 65% (FIG. 8C). These reductions in palmitateincorporation correlate well with the previously reported observationthat 20:5 is slightly more potent than 20:4 in inhibiting T cellsignaling and in displacing Fyn and Lck from detergent resistantmicrodomains (37). In the same report, the polyunsaturated fatty aciddocosahexaenoic acid (22:6) was less active than 20:4 and 20:5, and onlymoderately inhibited Fyn/Lck displacement from detergent resistantmicrodomains and TCR signaling. In agreement with these findings, 22:6was 10-20% less potent than 20:4 and 20:5 at inhibiting Fynpalmitoylation (data not shown).

Whether 20:4 and 20:5 inhibited localization of Fyn to detergentresistant microdomains in COS-1 cells was examined next. Cells weretreated with or without polyunsaturated fatty acids as described aboveand layered on the bottom of a sucrose gradient as described above.Fractions were collected and analyzed by immunoblotting with anti-Fynantibody.

As depicted in FIG. 8D, in untreated cells, 30% of Fyn localized todetergent resistant microdomains, in agreement with previous findings(47). Treatment with polyunsaturated fatty acids markedly reduced theability of Fyn to localize to detergent resistant microdomains, withonly 16% of Fyn found in detergent resistant microdomains in 20:4treated cells, and 5.3% in 20:5 treated cells. These finding clearlydemonstrate that the displacement of Fyn from detergent resistantmicrodomains is likely due to a polyunsaturated fatty acid-inducedreduction in Fyn palmitoylation.

Discussion

2-bromopalmitate inhibits Fyn fatty acylation in COS-1 cells

The ability of Src family members Fyn and Lck to participate insignaling through the T-cell receptor is critically dependent on theirfatty acylation with myristate and palmitate. In this study,2-bromopalmitate was identified as an inhibitor of Fyn fatty acylationand membrane targeting. This was accomplished by screening palmitateanalogs for their ability to inhibit incorporation of myristate andpalmitate into Fyn in transiently transfected COS-1 cells. Thisinhibition results in decreased membrane binding and localization todetergent resistant microdomains. Moreover, 2-bromopalmitate inhibitsfatty acylation and localization of Fyn, Lck and LAT to detergentresistant microdomains in Jurkat T cells. Consequently, this results inimpaired signaling through the T-cell receptor, as shown by a reductionin tyrosine phosphorylation, calcium flux and activation of the MAPkinase pathway. Furthermore, polyunsaturated fatty acids arachidonicacid (20:4) and eicosapentaenoic acid (20:5) are specific inhibitors ofFyn palmitoylation and localization to detergent resistant microdomainsin COS-1 cells. This may account for the ability of these compounds toinhibit T cell signaling as reported previously (37), and may be amechanism by which these agents exert their immunosuppressive andanti-inflammatory effects.

Protein palmitoylation occurs within an N-terminal myr-gly-cys motif,and that this event is dependent on myristoylation (12,13). The abilityof 2-bromopalmitate to partially inhibit myristoylation likely accountsfor some of the reduction in palmitoylation. However, the extent ofinhibition by 2-bromopalmitate on Fyn palmitoylation is always greaterthan that on myristoylation, implying that 2-bromopalmitate hasadditional, direct effects on palmitoylation (FIGS. 1A, 4A). A directeffect on palmitoylation is also supported by the observation that2-bromopalmitate inhibits membrane localization of a GAP43(10)-Fynconstruct, which is palmitoylated but not myristoylated (FIG. 3B).Furthermore, 2-hydroxymyristate, a known inhibitor of myristoylation(40,41), inhibits membrane localization of Fyn and Fyn(16)-eGFP to agreater extent than 2-bromopalmitate (FIG. 2B). This implies that2-bromopalmitate treated cells contain a population of myristoylated,non-palmitoylated Fyn that has a greater affinity for membranes thannon-acylated Fyn. Finally, the membrane localization of Fyn in thepresence of 2-bromopalmitate resembles that of the myristoylated,non-palmitoylated C3,6S Fyn mutant previously studied (13). Thus,2-bromopalmitate is an inhibitor of protein fatty acylation with somespecificity for palmitoylation.

Two possible mechanisms may account for the inhibitory effect of2-bromopalmitate on palmitoylation. One possibility is that2-bromopalmitate binds to PAT, but because of the steric bulk of thebromine, it cannot be transferred to the acceptor protein.Alternatively, 2-bromopalmitate may serve as a substrate for PAT. Inthis case, Fyn would be S-acylated with 2-bromopalmitate, buthydrophilic and steric effects of the bromine atom would reduce theprotein's affinity for membranes. In the absence of a radiolabeled formof 2-bromopalmitate, it is not possible at this point to distinguishbetween these two possibilities.

2-bromopalmitate Inhibits Fyn Fatty Acylation and Signaling in Jurkat TCells

The experiments depicted in FIG. 4 indicate that 2-bromopalmitateinhibits Fyn fatty acylation and localization to detergent resistantmicrodomains in Jurkat T cells. As a result, there is a marked reductionin tyrosine phosphorylation of key signaling molecules in CD3 stimulatedcells (FIG. 5), suggesting that signaling via the TCR is impaired.Interestingly, some proteins show an increase in the level ofphosphorylation in 2-bromopalmitate treated cells as compared to controlcells, even in the absence of CD3 stimulation. While the basis for thisbasal activation is unknown, it does not seem to be related to TCRactivation, since there is no effect on Ca⁺² flux or activation of MAPkinase pathway in unstimulated 2-bromopalmitate treated cells. If thisbasal activation is taken into account, then the reduction of tyrosinephosphorylation on the signaling molecules examined ranges from 70-100%(FIG. 5B).

PUFAs inhibit Fyn palmitoylation and localization to DRMs in COS-1 cells

Polyunsaturated fatty acids modulate immune responses by affecting Tcell function (48). Therefore these agents (particularly the n-3 series)have found clinical applications in the treatment of variousinflammatory diseases such as rheumatoid arthritis and Crohn's diseaserelapses (33,35,36) and as immunosuppressive agents (32). Despite thebroad clinical use of polyunsaturated fatty acids, the mechanism ofpolyunsaturated fatty acid-induced T cell inhibition had not beenelucidated. Recently, it was reported that the polyunsaturated fattyacid-induced inhibition of T cell activation is due to displacement ofSrc family kinases from the cytoplasmic layer of the detergent resistantmicrodomains (37). This displacement was hypothesized to be mediated bymodification of the DRM structure and composition. Here it was shownthat the exclusion of Src family kinase Fyn from detergent resistantmicrodomains in polyunsaturated fatty acid-treated cells is due toinhibition of palmitoylation.

In contrast to the saturated inhibitor 2-bromopaImitate, polyunsaturatedarachidonic acid (20:4) and eicosapentaenoic acid (20:5) have almost noeffect on Fyn myristoylation, and are rather specific forpalmitoylation. Several lines of evidence suggest that the mechanism ofinhibition of palmitoylation involves the use of polyunsaturated fattyacids as alternative substrates for S-acylation to Fyn. First, studiesof partially purified preparations of PAT reveal that longer chain fattyacyl CoAs, including stearate (18:0) and arachidonate (20:4) can competewith palmitate for incorporation into Fyn and Gαo (26,27). Secondly, Gαsubunits, P-selectin, asialoglycoprotein receptor and several plateletproteins have been shown to be S-acylated with stearate, arachidonateand eicosapentaenoate, in addition to palmitate (49,50). These resultsindicate that the fatty acid specificity of PAT and S-acylation is loosein vivo and in vitro. Thirdly, Fyn localization to the plasma membranefraction is not affected by polyunsaturated fatty acids (data notshown), even though incorporation of ¹²⁵I-IC16 is markedly reduced.Since myristoylation alone is not sufficient for stable membranebinding, it is likely that Fyn becomes dually fatty acylated byN-myristoylation and S-acylation with a polyunsaturated fatty acid. Thepresence of myristate and polyunsaturated fatty acid at the N-terminusof Fyn would provide strong affinity for binding to a membrane bilayer.However, the presence of a polyunsaturated, bulky acyl chain in thepolyunsaturated fatty acid would preclude specific localization to DRMSwhich, due to their liquid ordered domain structure, provide a localenvironment conducive to insertion of saturated, but not unsaturatedfatty acid chains (18).

In conclusion, specific fatty acids and fatty acid analogs function asinhibitors of protein fatty acylation and TCR mediated signaling. Theadvantage of using inhibitors that interfere with subcellularlocalization of a key protein is that it allows one to study signalingby endogenous cellular proteins and eliminates the need to overexpressmutant proteins. Thus, 2-bromopalmitate can be used as a powerful toolto study the role of Src kinases in the endogenous T cell signalingsystem, and may provide insight into the role of signaling in the onsetof disease. PUFA-induced inhibition of T cells is likely due to theinhibition of Src kinase palmitoylation. Though these agents arecurrently used in the clinic, their mechanism of action is still largelyunknown. A novel mechanism, inhibition of protein palmitoylation, mayaccount for the abilities of polyunsaturated fatty acids to treat orprevent a broad range of immune-based diseases.

The following references were cited herein:

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Any patents or publications mentioned in this specification areindicative of the levels of those skilled in the art to which theinvention pertains. These patents and publications are hereinincorporated by reference to the same extent as if each individualpublication was specifically and individually indicated to beincorporated by reference.

One skilled in the art will readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. The presentexamples along with the methods, procedures, treatments, molecules, andspecific compounds described herein are presently representative ofpreferred embodiments, are exemplary, and are not intended aslimitations on the scope of the invention. Changes therein and otheruses will occur to those skilled in the art which are encompassed withinthe spirit of the invention as defined by the scope of the claims.

1. A method of inhibiting Fyn/Lck fatty acylation and proteinpalmitoylation in a cell In an individual having autoimmune diseasecomprising: administering to said individual a pharmacologicallyeffective dose of 2-bromopalmitate.
 2. The method of claim 1, whereinsaid 2-bromopalmitate is administered in a dose of about 0.1 mg/kg toabout 100 mg/kg of total body weight of said individual.
 3. The methodof claim 1, wherein said 2-bromopalmitate inhibits proteinpalmitoylation within the N-terminus of the proteins.
 4. The method ofclaim 1, wherein said 2-bromopalmitate further inhibits myristoylationof proteins.
 5. The method of claim 1, wherein inhibiting Fyn/Lck fattyacylation further inhibits T cell signaling events.
 6. The method ofclaim 1, wherein said autoimmune disease is rheumatoid arthritis,Crohn's disease, diabetes, multiple sclerosis or systemic lupuserythematosus.
 7. A method of inhibiting T-cell receptor mediatedsignaling events in an individual having an autoimmune diseasecomprising: administering to said individual a pharmacologicallyeffective dose of 2-bromopalmitate; wherein 2-bromopalmitate inhibitsFyn/Lck fatty acylation in the T-cells thereby inhibiting T-cellreceptor mediated signaling events in the individual.
 8. The method ofclaim 7, wherein said 2-bromopalmitate is administered in a dose ofabout 0.1 to about 100 mg/kg of total body weight of said individual. 9.The method of claim 7, wherein said autoimmune disease is rheumatoidarthritis, diabetes, Crohn's disease, multiple sclerosis or systemiclupus erythematosus.